Project Summary A detailed understanding of the anatomical and molecular architectures of brain cells and their brain-wide organization is essential for interrogating human brain function and dysfunction. Extensive efforts have been made toward mapping brain cells through various lenses, which have established invaluable databases yielding new insights. However, integrative extraction of the multimodal properties of various cell-types brain-wide within the same brain, crucial to elucidating complex intercellular relationships, remains nearly impossible. We have developed high-throughput, cost-effective technology platforms to create a fully integrated three-dimensional (3D) human brain cell atlas by simultaneously mapping high-dimensional features (e.g., spatial, molecular, morphological, and microenvironment information) of all cells acquired from the same whole brain. The proposed work will establish the most comprehensive 3D human brain map to date, with unprecedented resolution and completeness. We envision that this atlas will facilitate the integration of a broad range of studies and allow the research community to interrogate human brain structure and function at multiple levels. In Aim 1, we will apply a novel technology to transform whole human brain tissue into indestructible hydrogel?tissue hybrids that allow highly multiplexed molecular labeling and subcellular-resolution volume imaging. In Aim 2, we will apply scalable labeling and imaging technologies to map the brain-wide 3D distribution of various cell-type and structural markers at subcellular resolution within the same brain. Our chemical engineering?based approach to this aim will enable cost-effective, lossless 3D labeling of the entire human brain at lower cost as traditional subsampling approaches. True volume labeling and subcellular-resolution imaging will allow us to extract fine morphological and connectivity information from labeled cells and reconstruct the microenvironment of all cells. In Aim 3, we will use a host of rapid and highly automated algorithms to perform unbiased, integrative high- dimensional phenotyping of all cells based on their spatial location, molecular expression, morphology, and microenvironment. In Aim 4, we will perform super-resolution phenotyping of cells in a selected brain region from the same sample used in Aim 3 to map inter-areal axonal connectivity at single-fiber resolution and to characterize chemical synapses. This integrative approach will likely unveil unique cell-types and brain regions, a crucial step toward a better understanding of brain function. The complete 3D dataset will be linked to magnetic resonance and diffusion spectrum images and existing reference atlases to facilitate the integration of a wide breadth of study at multiple levels and to make the data publicly accessible for mining and analysis.